Disassembly of the cone-shaped HIV-1 capsid after virus-cell fusion is a prerequisite for establishing a life-long infection. This step in HIV-1 entry, referred to as uncoating, is critical yet poorly understood. We have recently developed a novel strategy to visualize HIV-1 uncoating that is based on a fluorescently tagged oligomeric form of a capsid-binding host protein cyclophilin A (CypA-DsRed). CypA-DsRed, which is specifically packaged into virions through the high-avidity binding to the HIV-1 capsid, does not compromise the infectivity. This probe remains associated with cores after virus-cell fusion and is released upon uncoating. Supporting this notion is our finding that the rate of CypA-DsRed loss from individual post-fusion cores is modulated by mutations affecting the core stability and is accelerated by reverse transcription. The CypA-DsRed based imaging assay revealed a biphasic kinetic of HIV-1 uncoating, with a large number of cores shedding the capsid protein shortly after fusion. This assay also revealed marked differences in the uncoating phenotype in HeLa-derived cells and primary human macrophages. We propose to: (1) delineate CypA-DsRed interactions with HIV-1 core through functional and structural studies of CypA-DsRed/capsid complexes; (2) elucidate the relationship between reverse transcription and single core uncoating; and (3) investigate regulation of HIV-1 uncoating by host factors, such as Nup358 and CPSF6. These studies, employing a combination of genetic, biochemical and advanced imaging tools, are expected to provide critical insights into the dynamics and regulation of HIV-1 uncoating in living cells.